Copying of polynucleotides, more particularly amplification, is commonly used in molecular biology for studying, for example, the properties of genes. Problems in copying arise when the polynucleotide is damaged in some way.
By way of illustration, U.S. Pat. No. 5,035,996 describes a process for controlling contamination of polymerase chain reaction amplification reactions that uses the modified nucleotide, dUTP, in the amplification reaction. This process uses uracil DNA glycosylase (UDG) to eliminate those PCR products containing uracil to prevent contaminating subsequent PCR reactions. U.S. patent publication No. 2004-0067559 A1 also relies on modified bases in primer DNA prior to amplification and uses, for example, dUTP for incorporation into the amplicon. The amplicon can then be fragmented by adding, for example, UDG and Endonuclease (Endo) IV.
One amplification methodology referred to as hot start nucleic acid amplification has been used to lower mis-priming during polymerase chain reaction (PCR). In one type of hot start amplification, prevention of extension by the polymerase relies on the presence of a PCR primer with a blocked 3′ terminus in the PCR reaction (see for example U.S. Publication No. 2003-0119150). The primer is unblocked by a thermostable 3′-5′ exonuclease that is active at >37° C. Therefore, the polymerase will only extend the PCR primers once the exonuclease unblocks the 3′ end at >37° C. Alternatively the Taq polymerase is blocked and then activated at amplification temperatures.
Barnes, W. M. Proc. Natl. Acad. Sci. USA 91:2216-2220 (1994) describes the use of Vent® polymerase and Taq polymerase as an improvement over the use of Taq polymerase only in amplification. Ghadessy et al. reported a mutant Taq polymerase that is not halted by damaged or abasic sites (Ghadessy et al. Nature Biotechnol. 22(6):755-9 (2004)).
It has been reported that conventional amplification techniques are compromised if the DNA is substantially damaged (Di Bernardo et al. Nucl. Acids Res. 30:e16 (2002)). Degradation and/or fragmentation of DNA resulting from exposure to the environment and microorganisms which contain DNA nucleases is a frequent problem in forensics, diagnostic tests and routine amplification and affects fidelity and yield of the amplification product. In addition, the problem of degraded DNA is also faced by researchers who are analyzing the DNA obtained from extinct or extremely rare organisms that have been stored, frozen or fossilized.
Fromenty, B., et al. Nucl. Acids Res. 28(11):e50 (2000) and International Publication No. WO/0151656 reported that treatment with Exonuclease (Exo) III improved yields of long PCR. However, Fromenty also reported decreased yields of amplicon for DNA<500 bp when Exo III was used. One of the problems associated with the use of Exo III is that it degrades template and primers.
Di Benardo et al. Nucl. Acids Res. 30(4):e16 (2002) described the use of T4 DNA ligase (T4 ligase) and an E. coli polymerase as a pretreatment to amplify short regions of single-stranded DNA between cross-linked regions of double-stranded DNA.
Another approach to amplification of damaged DNA has been described in U.S. Publication No. 2003-0077581. Degraded nucleic acid was hybridized to non-degraded nucleic acid having a sequence homologous to the degraded nucleic acid. Regions of the degraded nucleic acid were then filled in with nucleotide precursors. The fragmented strands were then covalently linked using a polymerizing and/or ligating enzyme.
Preparations for improving amplification of damaged DNA can be obtained commercially from Sigma, St. Louis, Mo. and Qbiogene, now MP Biomedicals, Irvine, Calif. Although the compositions of these preparations are not stated, it is assumed that Exo III is contained in the preparation.
Others report the use of a combination of E. coli DNA PolI and T4 ligase for pre-amplification repair (Pusch, et al., Nucl. Acids Res. 26:857 (1998)). However, according to Pusch et al., the pre-amplification product must be purified before initiation of amplification.